Studies aimed at understanding the regulation of keratinocyte-specific gene expression are likely to provide insights into epidermal differentiation as well as its disruption during neoplastic transformation. Although a variety of general transcription factors influence epidermal gene expression, there is little information regarding the existence of trans-acting "master regulators" of keratinocyte differentiation analogous to Myo-D and its relatives in skeletal muscle. Treatment of cultured cells with 5'-bromo-2'-deoxyuridine (BrdU) for several cell divisions reversibly blocks differentiation of multiple cell types including chondrocytes, mammary epithelial cells, retinal pigment cells, erythroblasts, and skeletal myoblasts. The inhibition of differentiation occurs at BrdU concentrations that have minimal effects on proliferation and overall protein synthesis and is reversible: cells permitted to recover in medium without BrdU re-acquire the ability to terminally differentiate. Suppression of myogenesis by BrdU is associated with inhibition of Myo-D expression, and muscle-specific gene expression is restored in BrdU-treated myoblasts following introduction of a Myo-D expression vector. The suppression of differentiated functions by BrdU in other cell types may similarly be due to selective inhibition of critical regulatory molecules, potentially making this agent a novel tool for studying the regulation of keratinocyte differentiation. We have demonstrated that treatment of primary keratinocytes with BrdU blocks the expression of multiple epidermal differentiation markers in a time- and dose-dependent manner. Markers expressed both at early and late stages of epidermal differentiation were blocked by BrdU; in contrast, expression of keratins normally detected in embryonic epidermis was reactivated. Inhibition of differentiation-specific gene expression was also demonstrated using a reporter plasmid containing an epidermal-specific promoter, supporting the concept that BrdU influences terminal differentiation by altering the abundance or function of critical trans-acting factors.